Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations

Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N’Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126–54.00 μg/ml or LC95 × 3 = 0.7437–17.82 μg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

Dieldrin cross-resistance testing. To confirm the resistance profiles of both An. arabiensis colonies, initial WHO susceptibility tests were performed on An. arabiensis SENN (dieldrin-resistant) and An. arabiensis KGB (dieldrin-susceptible). A total of 115 KGB individuals were exposed to the discriminating concentration of dieldrin (0.4%) with 100% mortality observed after 60 min (Fig. 3A); demonstrating that this strain was susceptible to dieldrin.
Average 24-h mortality of 112 An. arabiensis SENN tested using 0.4% dieldrin impregnated filter papers was 12.1% (95% CI 1.17%-22.9%) (Fig. 3B). For the 107 An. arabiensis SENN mosquitoes tested with 4% dieldrin impregnated filter papers (10X discriminating concentration), average 24-h mortality was 16.5% (95% CI 1.33-31.73) (Fig. 3B); confirming that this strain was highly resistant to dieldrin. All An. arabiensis SENN which survived 4% dieldrin exposure possessed the A296S mutation. All An. arabiensis SENN tested in dieldrin bioassays were confirmed as An. arabiensis by species-specific PCR.  50 , indicating an absence of cross-resistance between broflanilide and dieldrin. A subset of An. arabiensis SENN (n = 290) tested against different broflanilide concentrations were screened for the presence of rdl A296S to investigate the potential for this resistance mechanism to mediate cross-resistance against broflanilide. There was no association between rdl A296S genotype and survival or death following exposure to any broflanilide concentration (Fisher's exact test = 0.6019). All An. arabiensis SENN screened for rdl A296S were confirmed as being An. arabiensis by species-specific PCR.

Discussion
The development of novel insecticide formulations for IRS whose efficacies are not compromised by pre-existing cross-resistance in vector populations, is crucial to sustain current gains in malaria vector control 33 . This multicentre study builds upon initial broflanilide DC testing performed with single insecticide-susceptible insectary colonies, using six mosquito strains, representing major Anopheles species; An. gambiae and An. arabiensis are sympatric malaria vectors across sub-Saharan Africa 34 , An. coluzzii is a pervasive malaria vector species in West Africa 35 and An. stephensi is the primary urban vector species in the Indian subcontinent 36 , which has become an invasive rural species in the Horn of Africa 37 and has recently been detected in Nigeria 38 . Study results demonstrated significant heterogeneity in mortality-dose responses following broflanilide exposure, between Anopheles species (e.g. An. stephensi SK vs. An. gambiae Kisumu KCMUCo), within Anopheles species (e.g. An. arabiensis KGB vs. An. arabiensis SENN) and even between the same insectary strain maintained at different testing facilities (An. gambiae Kisumu KCMUCo vs. An. gambiae Kisumu NIMR). Across all vector species tested, the ranges of DC generated by this study were 1.126 μg/ml to 54.00 μg/ml (LC 99 × 2) or 0.7437 μg/ml to 17.82 μg/ml (LC 95 × 3). These estimates provide an initial benchmark for broflanilide susceptibility monitoring, as part of ongoing VECTRON T500 community trials in Tanzania and Benin 21 . Further studies will be required on different Anopheles species and populations in order to identify what will be the definitive DC for broflanilide susceptibility monitoring in conjunction with the use of VECTRON T500 in malaria vector control programmes.
Our  20 with An. gambiae Kisumu CREC. The difference between the DCs in these studies may be explained by a difference in the method for coating bottles used in bioassay testing. In the current study, technical grade broflanilide was dissolved in acetone with 800 ppm Mero® (81% rapeseed oil methyl ester), as recommended by the commercial manufacturer. Although the role of Mero® in the pickup and uptake of some insecticides is not yet fully understood, it is known that it prevents insecticide crystallization, which can inhibit absorption across the insect cuticle, allowing broflanilide to remain in an amorphous state throughout bioassay testing. The addition of Mero®, therefore, increases the efficacy of broflanilide in bottle bioassays with mosquitoes, i.e., it decreases the concentration of broflanilide needed for lethality. Similarly, the efficacy of clothianidin in bottle bioassays has also been shown to be enhanced by the inclusion of Mero® when coating bottles 39 .
The differences between bioassay results using the same mosquito strain (An. gambiae Kisumu) maintained at two separate testing facilities (KCMUCo and NIMR) raises some interesting questions regarding direct comparability of insectary colony data. Differences in mosquito rearing conditions, including larval rearing conditions (e.g. crowding, access to nutrition) 40 , time of testing (e.g. night or day) 41,42 , temperature and humidity 43 , mosquito age 44 and physiological status 45 can have a significant effect on observed bioassay mortality. Whilst every effort is made to maintain standardized test conditions, according to WHO protocols 30 , even differences of 4 °C during holding periods can have a significant effect on mosquito mortality 43 , with lower temperatures associated with reduced mortality. Finally, an unascertainable amount of variation between the An. gambiae Kisumu strains maintained at different testing facilities may be attributable to long-term genetic divergence, and in turn, differences in relative colony fitness since these mosquito populations have been maintained in separate facilities for more than a decade. These observations support periodic in-depth strain characterization at both phenotypic and genotypic levels, as has been reported for recently colonized insecticide-resistant colonies 46,47 , to strengthen future laboratory screening of new insecticides.
A secondary objective of this study was to investigate whether there was any biological basis for crossresistance to broflanilide via the A296S mutation in the GABA-gated chloride receptor leading to dieldrin resistance (rdl). Despite the ban of dieldrin decades ago, the A296G and A296S rdl mutations have persisted in some contemporary vector populations at high frequencies [48][49][50] . We observed no evidence for cross-resistance to broflanilide in this study supporting its deployment in areas of pre-existing rdl; indeed, lower concentrations of broflanilide were required to induce complete mortality of the dieldrin-resistant An. arabiensis SENN strain, compared to its susceptible counterpart, An. arabiensis KGB, which may in part be explained by the fitness costs associated with highly insecticide-resistant populations, as shown in previous field studies 51,52 . This was further reinforced by a lack of association between the A296S rdl mutation and the outcomes of broflanilide bottle bioassays. Three additional amino acids, which surround the broflanilide binding pocket in the GABA receptor, have been identified that can disrupt insecticide binding: G331, I272 and L276 28 . Screening of the Ag1000 genome data has failed to identify any naturally-occurring mutations in these amino acids in Anopheles field populations 28 . These genetic regions warrant inclusion in newly developed amplicon-sequencing panels 53 , which are being rolled out to monitor insecticide resistance across Anopheles vector populations, in conjunction with standard insecticide susceptibility monitoring.
The variability in mortality-dose response to broflanilide, evidenced in this study, strongly advocates for further broflanilide DC testing, using additional insecticide-susceptible Anopheles colonies, particularly an An. funestus strain; this vector species predominates across southern sub-Saharan Africa and plays an increasing role in malaria transmission in areas where other vector species have been controlled by insecticidal interventions 54  www.nature.com/scientificreports/ Unfortunately, this was not feasible for inclusion in this multi-centre study, due to notorious difficulties rearing this particular species under controlled insectary conditions 55 . Broflanilide testing using wild Anopheles populations is also needed to demonstrate the efficacy of this novel insecticide to control pyrethroid-resistant vectors and to assess variability in the tolerance of these populations to broflanilide. Previous laboratory studies have demonstrated a lack of cross-resistance between mechanisms of resistance possessed by pyrethroidresistant insectary strains and broflanilide 20,28 . However, a plethora of complex coinciding, insecticide resistance mechanisms can be found in natural Anopheles populations 56-61 , which are not adequately reflected in genetically homogenous insectary colonies.

Conclusions
This multi-centre study, using six Anopheles insectary colonies, representing major malaria vector species, determined the putative discriminating concentration for broflanilide to range between LC 99 × 2 = 1.126 to 54.00 μg/ ml or LC 95 × 3 = 0.7437 to 17.82 μg/ml. Comparison of the susceptibility of dieldrin-resistant and -susceptible An. arabiensis colonies provided no phenotypic or genotypic evidence for cross-resistance to broflanilide via the A296S rdl mutation in the GABA-gated chloride receptor leading to dieldrin resistance. Use of the adjuvant Mero® increased broflanilide efficacy, highlighting the need to standardize bottle bioassay testing for this new insecticide. Differences in bioassay results using the same mosquito strain (An. gambiae Kisumu) maintained at two separate facilities raised issues regarding direct comparability of insectary colony data and emphasizes the need for periodic in-depth strain characterization to strengthen future laboratory screening of new insecticides.
Our study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.  68 . This strain has been exposed to dieldrin and confirmed resistant due to the GABA-gated chloride receptor mutation (Ala296Ser). Further characterization of the latter two strains is described below. In all three testing facilities, all life-cycle stages of colony mosquito populations were maintained under standard insectary conditions (25-27 °C, 80% relative humidity, light:dark cycles of 12-h each). In LSHTM mosquito larvae were reared in large white trays, with 12-h light-dark cycles, and fed NISHIKOI staple fish food pellets (Nishikoi, UK). In KCMUCo and NIMR, mosquito larvae were reared in large white round bowls, with 12-h light-dark cycles, and fed with TetraMin (Tetra, U.S.).

Mosquito strains. Six
Adult mosquitoes were kept in cages of ~ 30 × 30 × 30 cm at varying densities, with 10% glucose provided ad libitum. In LSHTM, colony cages were maintained by regular blood feeding using a Hemotek feeder. In KCMUCo and NIMR, colony cages were maintained by regular blood feeding on Guinea Pigs.
Broflanilide discriminating concentration testing. A discriminating concentration is defined as the concentration of insecticide that in a standard period of exposure, is used to discriminate the proportions of susceptible and resistant phenotypes in a sample of a mosquito population 29 . Discriminating concentration testing of broflanilide was undertaken using the CDC bottle bioassay method, but with minor modifications to the published guidelines (Fig. 1) 64 . Probit analysis was used to determine thirteen concentrations of broflanilide for testing (100, 46.4, 21.5, 10, 4.6, 2.2, 1, 0.46, 0.22, 0.1, 0.046, 0.022 and 0.01 μg/ml). Technical grade broflanilide (Mitsui Agro, Inc., Japan) was dissolved in acetone with 800 ppm Mero®; the adjuvant Mero® was used to ensure the insecticide was distributed evenly throughout each bottle and to prevent crystallisation of broflanilide during the conduct of bioassays. Control bottles consisting of acetone alone and acetone + 800 ppm Mero® were run in parallel during each bioassay.
Each Wheaton 250 ml bottle and cap was coated using 1 ml of insecticide solution by rolling it and inverting the bottle. In parallel, control bottles were coated with either 1 ml acetone or 1 ml acetone + 800 ppm Mero® per bottles. Once coated, all bottles were covered with a cotton sheet and left to dry in the dark overnight; and Individual mosquitoes were identified to species-level using species-specific PCR primers for An. gambiae s.s. and An. arabiensis (Table 3) 63 . Each 20 μl reaction contained 20-40 ng of gDNA, 10 μl HotStart Taq 2X Master Mix (New England Biolabs, UK) and 25 pmol/ml of primers AR-3T, GA-3T and IMP-UN. Prepared reactions were run on a BioRad T100™ thermal cycler with the following conditions: 95 °C for 5 min, followed by 30 amplification cycles (95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s) and a final elongation step at 72 °C for 5 min. PCR products were visualised on 2% E-gel agarose gels in an Invitrogen E-gel iBase Real-Time Transilluminator. A Quick-Load® 100 bp DNA ladder (New England Biolabs, UK) was used to determine band size. PCR products of 387 bp or 463 bp were indicative of An. arabiensis or An. gambiae s.s., respectively, relative to positive controls; no-template negative controls were included with all reaction runs.
The presence of the A296S rdl mutation in An. arabiensis was determined using a TaqMan assay 70 . Each 20 μl reaction contained 20-40 ng of gDNA, 10 μl 2X PrimeTime® Gene Expression Master Mix (Integrated DNA Technologies, USA), 800 nM of primers SerRdlF and SerRdlR and 200 nM of probes WT2 and Ser (Table 3). Prepared reactions were run on a Stratagene Mx3005P qPCR system with the following conditions: 95 °C for 10 min, followed by 40 amplification cycles (95 °C for 10 s, 60 °C for 45 s), and lastly a dissociation curve. Notemplate negative controls were included with all reaction runs. The presence of a wild-type individual was indicated by a substantial increase in HEX signal, the presence of the A296S rdl mutation was indicated by a substantial increase in FAM signal; increase in both signals indicated a heterozygote.

Data analysis. Discriminating concentration (DC) determination was undertaken using BioRssay 71 in
RStudio v4.0.2 72 . Mortality-dose regression analysis using a generalized linear model was performed per mosquito strain. Lethal doses for 50%, 95% and 99% (LC 50 , LC 95 and LC 99 ) with 95% confidence intervals were calculated. The LC 95 value was multiplied by three to determine the DC as per the Lees et al. method 31 . The DC was also calculated by multiplying the LC 99 by two as per the WHO approach 32 . Differences in dose-mortality responses between strains were evaluated using pair-wise comparisons with Bonferroni correction. All other statistical analyses were conducted in GraphPad Prism 9.4.0.
Ethics approval. Ethical approval for the study was obtained from the London School of Hygiene and Tropical Medicine (LSHTM; ref#26035) and the National Institute for Medical Research (NIMR) in Tanzania (NIMR/HQ/R.8a/VOL.IX/3520). KCMUCo and NIMR obtained approval from the Animal Welfare and Ethical Review Board of LSHTM (ref#2019-14) for use of animals for mosquito maintenance. Study procedures and reporting are in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All study procedures were performed in accordance with relevant guidelines and regulations.  IMP-UN  GCT GCG AGT TGT AGA GAT GCG   GA-3T  GCT TAC TGG TTT GGT CGG CATGT   AR-3T  GTG TTA AGT GTC CTT CTC CGTC   A296S rdl detection   SerRdlF  TCA TAT CGT GGG TAT CAT TTT GGC TAAAT   SerRdlR  TCG TTG ACG ACA TCA GTG TTGT   WT2  /HEX/TTA  www.nature.com/scientificreports/

Data availability
The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.